Hepatitis C Virus (HCV) accounts for nearly all cases of non-A, non-B hepatitis (NANBH) (Choo, Q.-L., et al., Proc. Natl. Acad. Sci. USA 88: 2451-2455 (1988)) and is a persistent health threat worldwide, with more than one million new cases reported annually (Zein, N. N. Clin. Micro. Rev. 13: 223-235 (2000)). HCV infection is almost always chronic and persistent. The most severe consequences of HCV infection are chronic liver disease and death, and HCV infection is the primary impetus for liver transplantation in the US (Zein, supra).
HCV is a positive strand single-stranded RNA virus approximately 10 kb long belonging to the Flaviviridae family (Zein, supra). There is considerable heterogeneity among isolates found in different geographic regions. These differences have been classified into multiple genotypes and subtypes. Although various different criteria have been used to characterize these genotypes, two principal modes of classification have been adopted. The more widely used of these was created by Peter Simmonds and uses Arabic numerals to denote different genotypes and latin letters for subtypes, e.g. type 1a, 1b, 2a, etc. (reviewed in Simmonds, P. Hepatology. February; 21(2):570-83 (1995) and Simmonds, P. J Hepatol.; 31 Suppl 1:54-60 (1999)). According to this system, genotypes 1-3 are the prevalent types found in North America, Europe, and Japan, and the remaining types are found at various frequencies in parts of Asia and Africa. Thus in some instances HCV genotype may be of epidemiological importance, for example in determining the etiology of infection.
Efforts have been undertaken to elucidate the clinical significance of different genotypes. Some studies suggest that infections of type 1, in particular type 1b, may be associated with more severe disease and earlier recurrence (Zein, N. N. et al., Liver Transplant. Surg. 1: 354-357 (1995); Gordon et al., Transplantation 63: 1419-1423 (1997)). Certain studies have also indicated that genotypes other than type 1 (e.g. 1a or 1b) may respond more favorably to various treatments, e.g. interferon (McHutchison, J. G., et al., N. Engl. J. Med., 339: 1485-1492 (1998)). It has been suggested that determination of HCV genotype in combination with other diagnostic markers, such as viral load, may be of value in arriving at disease prognoses (Zein, N. N. supra), and determining the course of treatment (National Institutes of Health Consensus Development Conference Statement; Management of Hepatitis C: 2002; Jun. 10-11, 2002).
Different regions of the HCV genome have been used to determine genotype. The HCV genome includes relatively conserved regions, such as the 5′ and 3′ untranslated regions (UTR), variable regions (e.g. E1 and non-structural (NS) 5B), as well as hypervariable regions such as those encoding the envelope proteins (Halfon, P. CLI, April 2002). Studies have been carried out to correlate the presence of particular sequences in the conserved regions with sequences in the variable regions, in particular the NS-5B (Stuyver, L., et al., J. Clin. Micro., 34: 2259-2266 (1996)). As a result of such studies, genotyping assays based on conserved regions, particularly the 5′ UTR, have been developed to simplify the task of identifying which viral type or types are present in a specimen. Given the existence of commercially available viral load assays that rely on amplifying all or part of the 5′ UTR, the ability to determine HCV genotype based on discrete sequence differences in this conserved region presents a convenient means of obtaining extensive diagnostic information from a single amplified nucleic acid, e.g. a RT-PCR or Transcription Mediated Amplification (TMA) amplicon.
Various molecular biological methods have been applied to the task of determining HCV genotype using the 5′ UTR. These include reverse dot-blot analysis (e.g. Inno LIPA, Innogenetics, Ghent, Belgium, as described in Stuyver, L. et al., J Clin Microbiol. 1996 September; 34(9):2259-66, U.S. Pat. No. 6,495,670 and related U.S. and international patents and pending applications; direct DNA sequencing (e.g. TRUEGENE HCV 5′NC genotyping kit, Bayer Diagnostics, Berkeley, Calif., as described in Germer, J. J. et al. J Clin Microbiol. 2003 October; 41(10):4855-7), and pyrosequencing (Pyrosequencing AB, Uppsala, Sweden, as described in U.S. Pat. No. 6,258,568 and related U.S. and international patents and pending applications).
In addition to these molecular methods, serological methods for determining genotype have been introduced, e.g. the RIBA SIA test (Chiron Corp., Emeryville, Calif.) and the Murex HCV serotyping enzyme immune assay (Murex Diagnostics Ltd, Dartford, UK). Some studies indicate that serologic typing may be limited in terms of specificity and sensitivity (Zein, supra).
Therefore, there exists a need for a rapid, sensitive, accurate, and homogeneous method for accurately determining HCV genotype in a clinical sample, e.g. blood or blood fraction, without the need for electrophoretic or dot-blot techniques. Given the current reliance on molecular methods, it is likely that there will be an ongoing and increasing need for such scalable and automatable methods of determining HCV genotype.